Factor VIII (“FVIII”) is a blood plasma glycoprotein of about 280 kDa molecular mass. It is involved in the cascade of coagulation reactions that lead to blood clotting. The most common bleeding disorder is caused by a deficiency of functional Factor VIII, called haemophilia A. It is treated with replacement of Factor VIII, either plasma derived or recombinant. Factor VIII is used for acute and prophylactic treatment of bleedings in haemophilia A patients.
The amino acid sequence of Factor VIII is organized into three structural domains: a triplicated A domain of 330 amino acids, a single B domain of 980 amino acids, and a duplicated C domain of 150 amino acids. The B domain has no homology to other proteins and provides 18 of the 25 potential asparagine(N)-linked glycosylation sites of this protein. The B domain has apparently no function in coagulation. B-domain deleted Factor VIII molecules have unchanged procoagulant activity compared to full-length Factor VIII. Some recombinant Factor VIII (rFVIII) preparations are B-domain deleted.
In plasma, Factor VIII is stabilized by association with Von Willebrand Factor protein (“vWF”), which appears to inhibit clearance of Factor VIII e.g. by proteolysis or receptor-mediated clearance via the LRP-receptor. In circulation, Von Willebrand Factor is present in a 50-fold molar excess relative to Factor VIII under normal physiological conditions.
Von Willebrand Factor is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis, Von Willebrand Factor acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, Von Willebrand Factor serves as a carrier and stabilizing protein for procoagulant Factor VIII. Von Willebrand Factor is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The precursor polypeptide, pre-pro-Von Willebrand Factor, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in mature plasma Von Willebrand Factor (Fischer et al., FEBS Lett. 351: 345-348, 1994). Upon secretion into plasma, Von Willebrand Factor circulates in the form of various species with different molecular sizes. These Von Willebrand Factor molecules consist of oligo- and multimers of the mature subunit of 2050 amino acid residues. Von Willebrand Factor can be usually found in plasma as multimers ranging in size approximately from 500 to 20.000 kDa (Furlan, Ann Hematol. 1996 June; 72(6):341-8).
The average in vivo half-life of human Factor VIII in the human circulation is approximately 12 hours. Von Willebrand Factor might decrease possible immunoreactions against Factor VIII when in complex with Factor VIII by shielding FVIII from known potential inhibitor antibody sites on the heavy chain (A2 domain) and the light chain (A3/C2 domain) (Ragni, 3 Thromb. Haemost. 10: 2324-2327, 2012) or on other potential antibody inhibitor sites on the Factor VIII molecule.
A further bleeding disorder in humans is Von Willebrand's disease (vWD). Depending on the severity of the bleeding symptoms, vWD can be treated by replacement therapy with concentrates containing Von Willebrand Factor, in general derived from plasma but recombinant Von Willebrand Factor also is under development. Von Willebrand Factor is known to stabilize Factor VIII in vivo and, thus, plays a crucial role to regulate plasma levels of Factor VIII and as a consequence is a central factor to control primary and secondary haemostasis.
Until today, the standard treatment of Haemophilia A and vWD involves frequent intravenous infusions of preparations of Factor VIII and Factor VIII/Von Willebrand Factor concentrates. These replacement therapies are generally effective, however, for example in severe haemophilia A patients undergoing prophylactic treatment Factor VIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half life of Factor VIII of about 12 hours. Already by achieving Factor VIII levels above 1% of normal human plasma corresponding to a raise of Factor VIII levels by 0.01 U/ml, severe haemophilia A is turned into moderate haemophilia A. In prophylactic therapy, the dosing regime is designed such that the levels of Factor VIII activity do not fall below levels of 2-3% of the Factor VIII activity of non-haemophiliacs.
The administration of a Factor VIII via intravenous administration (i.v.) is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done in home treatment by the patients themselves or by the parents of children being diagnosed for haemophilia A. In addition, frequent intravenous injections inevitably result in scar formation, interfering with future infusions. Still, i.v. treatment might be needed in emergency situation or surgery, i.e. when a high Factor VIII-level is needed immediately.
Subcutaneous administration (s.c.) has been proposed for Factor VIII, e.g. in WO 95/01804 A1 and WO 95/026750 A1. However, very high doses of Factor VIII had to be administered to achieve an acceptable bioavailability.
Another approach to improve the bioavailability upon non-intravenous administration has been to use albumin-fused Factor VIII (WO 2011/020866 A2).
WO 2013/057167 A1 proposes to administer Factor VIII in combination with sulphated glycosaminoglycans via non-intravenous administration, optionally together with Von Willebrand Factor.
WO 2008/151817 A1 describes the general use of uncleaved Von Willebrand Factor multimers for stabilisation of Factor VIII, plasma derived or recombinant (full-length and deletion mutants) intended for extravascular treatment.
WO 2013/160005 A1 describes the general use of recombinant Von Willebrand Factor or recombinant Von Willebrand Factor-fragments to improve bioavailability after s.c. treatment for very specific Factor VIII molecules, wherein the said Factor VIII molecules comprise a truncated B domain at a size of 100-400 amino acids. According to WO 2013/160005 A1 Factor VIII molecules with truncated B domains between 100 and 400 amino acids have a higher Factor VIII bioavailability compared to Factor VIII having the entire B domain or B domain truncated Factor VIII molecules having no or only a few amino acids.
There is still a need for Factor VIII preparations showing improved bioavailability, stability and/or lower risk for antibody generation thereby avoiding drawbacks of prior art.
It is the object of the present invention to provide alternative Factor VIII preparations. Preferably, these preparations should show improved stability, improved bioavailability and/or reduced risk for immunological reactions.
In one embodiment, this object is achieved by a composition comprising a complex of Factor VIII and one or more Von Willebrand Factor peptides, wherein the Von Willebrand Factor peptides comprise at least the amino acids 764 to 1035 and 1691 to 1905 of SEQ ID No. 1 but not amino acids 2255 to 2645 of SEQ ID NO 1.
According to the present invention, a Factor VIII preparation comprising Von Willebrand Factor peptides is provided. Factor VIII form a complex with the comprising Von Willebrand Factor peptides.
Factor VIII as used herein covers full-length Factor VIII, B domain deleted Factor VIII or a Factor VIII wherein the B domain has been replaced by an artificial linker or a fragment of the natural B domain or a combination of both, i.e. the B-domain has a different size compared to full-length Factor VIII. It also covers Factor VIII with a limited number of modifications having insertion, deletion or substitutions, especially Factor VIII adapted to haplotypes as described in K. R. Viel, et al. New England J Med 2009; 360:1618-1627. Preferably, the sequence homology to Factor VIII (as defined in amino acids 20-2351 of P00451 of SwissProt Jul. 21, 1986) but disregarding the homology in the B-Domain of 99% according to FASTA as implemented in FASTA version 36, based on W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227-258. In other words, when calculating a sequence homology, the B-domain is not included in the comparison of both proteins. Also covered is modified Factor VIII, like HES-Factor VIII or PEG Factor VIII or Factor VIII Fc fusion proteins and Factor VIII albumin fusion proteins as described in Oldenburg, Haemophilia (2014), 20 (Suppl. 4), 23-28.
The Factor VIII of the present invention may be plasma derived or recombinant Factor VIII. When recombinant Factor VIII is used, it is preferably expressed in a human cell line to mimic human glycosylation pattern (Casademunt, Eur J Haematol. 2012; 89:165-76) or as described in WO 2010/020690.
Von Willebrand Factor peptides as used herein are peptides comprising at least amino acids 764 to 1035 of SEQ ID No. 1 and 1691 to 1905 of SEQ ID No. 1 in a single amino acid chain. These amino acids may be part of a longer sequence comprising both of these sequences together. In other words, the Von Willebrand peptides of the invention comprise both SEQ ID No. 5 and SEQ ID No. 6. They may comprise further parts of Von Willebrand Factor, excluding all the amino acids 2255 to 2645 (SEQ ID No. 7). The Von Willebrand peptides may comprise other sequences that are part of SEQ ID No. 1 or sequences that are not part of SEQ ID No. 1, e.g. amino acid linkers or the like. Preferably, the total amount of amino acids that are not part of SEQ ID No. 1 is not more than 50, not more than 20 or not more than 10 amino acids.
One important aspect of the invention is that amino acids 2255 to 2645 of SEQ ID No. 1 are not part of the Von Willebrand Factor peptides. In other words, the Von Willebrand Factor peptides do not comprise any sequence that has at least 90% homology to SEQ ID No. 7 according to FASTA, described below.
SEQ ID No. 1 is sequence P04275 of Swiss Prot database as of Jan. 11, 2011.
The Von Willebrand Factor peptides in the composition of the present invention may be peptides having the same sequence or may be a mixture of peptides having sequences as defined above.
Typically a molecular ratio of Factor VIII and Von Willebrand Factor peptides will be between 1:1 and 1:20, preferably 1:2 to 1:10. If the Von Willebrand factor peptides are in the form of dimers or multimers, the molecular ratio is calculated on a single amino acid chain, i.e. a complex of a Factor VIII molecule with a dimer of Von Willebrand factor peptides will have a ratio of 1:2.
A complex, as used herein refers to a non-covalent binding of Factor VIII to one or more Von Willebrand Factor peptides.
In a preferred embodiment of the invention, the Von Willebrand Factor peptides are fragments of Von Willebrand Factor, i.e. N-terminal and/or C-terminal truncated forms of Von Willebrand Factor.
In one embodiment, the fragments comprise amino acids 764 to 1905 of SEQ ID No. 1.
A further embodiment of the invention is a composition comprising a complex of Factor VIII and one or more Von Willebrand Factor peptides that are fragments of Von Willebrand Factor and have an amino acid sequence that corresponds to the amino acid sequence of SEQ ID NO 1 starting form amino acid 764 and ending between amino acid 1905 and 2153 with up to 20, or up to 10 modifications selected from amino acid deletions, amino acid insertions or amino acid substitutions.
Preferred Von Willebrand Factor peptides are:
Peptides having the sequence 764 to 1905 of SEQ ID No. 1
Peptides having the sequence 764 to 1906 of SEQ ID No. 1
Peptides having the sequence 764 to 1907 of SEQ ID No. 1
Peptides having the sequence 764 to 1908 of SEQ ID No. 1
Peptides having the sequence 764 to 1909 of SEQ ID No. 1
Peptides having the sequence 764 to 1910 of SEQ ID No. 1
Peptides having the sequence 764 to 1911 of SEQ ID No. 1
Peptides having the sequence 764 to 1912 of SEQ ID No. 1
Peptides having the sequence 764 to 1913 of SEQ ID No. 1
Peptides having the sequence 764 to 1914 of SEQ ID No. 1
Peptides having the sequence 764 to 1915 of SEQ ID No. 1
Peptides having the sequence 764 to 1916 of SEQ ID No. 1
Peptides having the sequence 764 to 1917 of SEQ ID No. 1
Peptides having the sequence 764 to 1918 of SEQ ID No. 1
Peptides having the sequence 764 to 1919 of SEQ ID No. 1
Peptides having the sequence 764 to 1920 of SEQ ID No. 1
Peptides having the sequence 764 to 1921 of SEQ ID No. 1
Peptides having the sequence 764 to 1922 of SEQ ID No. 1
Peptides having the sequence 764 to 1923 of SEQ ID No. 1
Peptides having the sequence 764 to 1924 of SEQ ID No. 1
Peptides having the sequence 764 to 1925 of SEQ ID No. 1
Peptides having the sequence 764 to 1926 of SEQ ID No. 1
Peptides having the sequence 764 to 1927 of SEQ ID No. 1
Peptides having the sequence 764 to 1928 of SEQ ID No. 1
Peptides having the sequence 764 to 1929 of SEQ ID No. 1
Peptides having the sequence 764 to 1930 of SEQ ID No. 1
Peptides having the sequence 764 to 1931 of SEQ ID No. 1
Peptides having the sequence 764 to 1932 of SEQ ID No. 1
Peptides having the sequence 764 to 1933 of SEQ ID No. 1
Peptides having the sequence 764 to 1934 of SEQ ID No. 1
Peptides having the sequence 764 to 1935 of SEQ ID No. 1
Peptides having the sequence 764 to 1936 of SEQ ID No. 1
Peptides having the sequence 764 to 1937 of SEQ ID No. 1
Peptides having the sequence 764 to 1938 of SEQ ID No. 1
Peptides having the sequence 764 to 1939 of SEQ ID No. 1
Peptides having the sequence 764 to 1940 of SEQ ID No. 1
Peptides having the sequence 764 to 1941 of SEQ ID No. 1
Peptides having the sequence 764 to 1942 of SEQ ID No. 1
Peptides having the sequence 764 to 1943 of SEQ ID No. 1
Peptides having the sequence 764 to 1944 of SEQ ID No. 1
Peptides having the sequence 764 to 1945 of SEQ ID No. 1
Peptides having the sequence 764 to 1946 of SEQ ID No. 1
Peptides having the sequence 764 to 1947 of SEQ ID No. 1
Peptides having the sequence 764 to 1948 of SEQ ID No. 1
Peptides having the sequence 764 to 1949 of SEQ ID No. 1
Peptides having the sequence 764 to 1950 of SEQ ID No. 1
Peptides having the sequence 764 to 1951 of SEQ ID No. 1
Peptides having the sequence 764 to 1952 of SEQ ID No. 1
Peptides having the sequence 764 to 1953 of SEQ ID No. 1
Peptides having the sequence 764 to 1954 of SEQ ID No. 1
Peptides having the sequence 764 to 1955 of SEQ ID No. 1
Peptides having the sequence 764 to 1956 of SEQ ID No. 1
Peptides having the sequence 764 to 1957 of SEQ ID No. 1
Peptides having the sequence 764 to 1958 of SEQ ID No. 1
Peptides having the sequence 764 to 1959 of SEQ ID No. 1
Peptides having the sequence 764 to 1960 of SEQ ID No. 1
Peptides having the sequence 764 to 1961 of SEQ ID No. 1
Peptides having the sequence 764 to 1962 of SEQ ID No. 1
Peptides having the sequence 764 to 1963 of SEQ ID No. 1
Peptides having the sequence 764 to 1964 of SEQ ID No. 1
Peptides having the sequence 764 to 1965 of SEQ ID No. 1
Peptides having the sequence 764 to 1966 of SEQ ID No. 1
Peptides having the sequence 764 to 1967 of SEQ ID No. 1
Peptides having the sequence 764 to 1968 of SEQ ID No. 1
Peptides having the sequence 764 to 1969 of SEQ ID No. 1
Peptides having the sequence 764 to 1970 of SEQ ID No. 1
Peptides having the sequence 764 to 1971 of SEQ ID No. 1
Peptides having the sequence 764 to 1972 of SEQ ID No. 1
Peptides having the sequence 764 to 1973 of SEQ ID No. 1
Peptides having the sequence 764 to 1974 of SEQ ID No. 1
Peptides having the sequence 764 to 1975 of SEQ ID No. 1
Peptides having the sequence 764 to 1976 of SEQ ID No. 1
Peptides having the sequence 764 to 1977 of SEQ ID No. 1
Peptides having the sequence 764 to 1978 of SEQ ID No. 1
Peptides having the sequence 764 to 1979 of SEQ ID No. 1
Peptides having the sequence 764 to 1980 of SEQ ID No. 1
Peptides having the sequence 764 to 1981 of SEQ ID No. 1
Peptides having the sequence 764 to 1982 of SEQ ID No. 1
Peptides having the sequence 764 to 1983 of SEQ ID No. 1
Peptides having the sequence 764 to 1984 of SEQ ID No. 1
Peptides having the sequence 764 to 1985 of SEQ ID No. 1
Peptides having the sequence 764 to 1986 of SEQ ID No. 1
Peptides having the sequence 764 to 1987 of SEQ ID No. 1
Peptides having the sequence 764 to 1988 of SEQ ID No. 1
Peptides having the sequence 764 to 1989 of SEQ ID No. 1
Peptides having the sequence 764 to 1990 of SEQ ID No. 1
Peptides having the sequence 764 to 1991 of SEQ ID No. 1
Peptides having the sequence 764 to 1992 of SEQ ID No. 1
Peptides having the sequence 764 to 1993 of SEQ ID No. 1
Peptides having the sequence 764 to 1994 of SEQ ID No. 1
Peptides having the sequence 764 to 1995 of SEQ ID No. 1
Peptides having the sequence 764 to 1996 of SEQ ID No. 1
Peptides having the sequence 764 to 1997 of SEQ ID No. 1
Peptides having the sequence 764 to 1998 of SEQ ID No. 1
Peptides having the sequence 764 to 1999 of SEQ ID No. 1
Peptides having the sequence 764 to 2000 of SEQ ID No. 1
Peptides having the sequence 764 to 2001 of SEQ ID No. 1
Peptides having the sequence 764 to 2002 of SEQ ID No. 1
Peptides having the sequence 764 to 2003 of SEQ ID No. 1
Peptides having the sequence 764 to 2004 of SEQ ID No. 1
Peptides having the sequence 764 to 2005 of SEQ ID No. 1
Peptides having the sequence 764 to 2006 of SEQ ID No. 1
Peptides having the sequence 764 to 2007 of SEQ ID No. 1
Peptides having the sequence 764 to 2008 of SEQ ID No. 1
Peptides having the sequence 764 to 2009 of SEQ ID No. 1
Peptides having the sequence 764 to 2010 of SEQ ID No. 1.
Peptides having the sequence 764 to 2011 of SEQ ID No. 1
Peptides having the sequence 764 to 2012 of SEQ ID No. 1
Peptides having the sequence 764 to 2013 of SEQ ID No. 1
Peptides having the sequence 764 to 2014 of SEQ ID No. 1
Peptides having the sequence 764 to 2015 of SEQ ID No. 1
Peptides having the sequence 764 to 2016 of SEQ ID No. 1
Peptides having the sequence 764 to 2017 of SEQ ID No. 1
Peptides having the sequence 764 to 2018 of SEQ ID No. 1
Peptides having the sequence 764 to 2019 of SEQ ID No. 1
Peptides having the sequence 764 to 2020 of SEQ ID No. 1
Peptides having the sequence 764 to 2021 of SEQ ID No. 1
Peptides having the sequence 764 to 2022 of SEQ ID No. 1
Peptides having the sequence 764 to 2023 of SEQ ID No. 1
Peptides having the sequence 764 to 2024 of SEQ ID No. 1
Peptides having the sequence 764 to 2025 of SEQ ID No. 1
Peptides having the sequence 764 to 2026 of SEQ ID No. 1
Peptides having the sequence 764 to 2027 of SEQ ID No. 1
Peptides having the sequence 764 to 2028 of SEQ ID No. 1
Peptides having the sequence 764 to 2029 of SEQ ID No. 1
Peptides having the sequence 764 to 2030 of SEQ ID No. 1
Peptides having the sequence 764 to 2031 of SEQ ID No. 1
Peptides having the sequence 764 to 2032 of SEQ ID No. 1
Peptides having the sequence 764 to 2033 of SEQ ID No. 1
Peptides having the sequence 764 to 2034 of SEQ ID No. 1
Peptides having the sequence 764 to 2035 of SEQ ID No. 1
Peptides having the sequence 764 to 2036 of SEQ ID No. 1
Peptides having the sequence 764 to 2037 of SEQ ID No. 1
Peptides having the sequence 764 to 2038 of SEQ ID No. 1
Peptides having the sequence 764 to 2039 of SEQ ID No. 1
Peptides having the sequence 764 to 2040 of SEQ ID No. 1
Peptides having the sequence 764 to 2041 of SEQ ID No. 1
Peptides having the sequence 764 to 2042 of SEQ ID No. 1
Peptides having the sequence 764 to 2043 of SEQ ID No. 1
Peptides having the sequence 764 to 2044 of SEQ ID No. 1
Peptides having the sequence 764 to 2045 of SEQ ID No. 1
Peptides having the sequence 764 to 2046 of SEQ ID No. 1
Peptides having the sequence 764 to 2047 of SEQ ID No. 1
Peptides having the sequence 764 to 2048 of SEQ ID No. 1
Peptides having the sequence 764 to 2049 of SEQ ID No. 1
Peptides having the sequence 764 to 2050 of SEQ ID No. 1
Peptides having the sequence 764 to 2051 of SEQ ID No. 1
Peptides having the sequence 764 to 2052 of SEQ ID No. 1
Peptides having the sequence 764 to 2053 of SEQ ID No. 1
Peptides having the sequence 764 to 2054 of SEQ ID No. 1
Peptides having the sequence 764 to 2055 of SEQ ID No. 1
Peptides having the sequence 764 to 2056 of SEQ ID No. 1
Peptides having the sequence 764 to 2057 of SEQ ID No. 1
Peptides having the sequence 764 to 2058 of SEQ ID No. 1
Peptides having the sequence 764 to 2059 of SEQ ID No. 1
Peptides having the sequence 764 to 2060 of SEQ ID No. 1
Peptides having the sequence 764 to 2061 of SEQ ID No. 1
Peptides having the sequence 764 to 2062 of SEQ ID No. 1
Peptides having the sequence 764 to 2063 of SEQ ID No. 1
Peptides having the sequence 764 to 2064 of SEQ ID No. 1
Peptides having the sequence 764 to 2065 of SEQ ID No. 1
Peptides having the sequence 764 to 2066 of SEQ ID No. 1
Peptides having the sequence 764 to 2067 of SEQ ID No. 1
Peptides having the sequence 764 to 2068 of SEQ ID No. 1
Peptides having the sequence 764 to 2069 of SEQ ID No. 1
Peptides having the sequence 764 to 2070 of SEQ ID No. 1
Peptides having the sequence 764 to 2071 of SEQ ID No. 1
Peptides having the sequence 764 to 2072 of SEQ ID No. 1
Peptides having the sequence 764 to 2073 of SEQ ID No. 1
Peptides having the sequence 764 to 2074 of SEQ ID No. 1
Peptides having the sequence 764 to 2075 of SEQ ID No. 1
Peptides having the sequence 764 to 2076 of SEQ ID No. 1
Peptides having the sequence 764 to 2077 of SEQ ID No. 1
Peptides having the sequence 764 to 2078 of SEQ ID No. 1
Peptides having the sequence 764 to 2079 of SEQ ID No. 1
Peptides having the sequence 764 to 2080 of SEQ ID No. 1
Peptides having the sequence 764 to 2081 of SEQ ID No. 1
Peptides having the sequence 764 to 2082 of SEQ ID No. 1
Peptides having the sequence 764 to 2083 of SEQ ID No. 1
Peptides having the sequence 764 to 2084 of SEQ ID No. 1
Peptides having the sequence 764 to 2085 of SEQ ID No. 1
Peptides having the sequence 764 to 2086 of SEQ ID No. 1
Peptides having the sequence 764 to 2087 of SEQ ID No. 1
Peptides having the sequence 764 to 2088 of SEQ ID No. 1
Peptides having the sequence 764 to 2089 of SEQ ID No. 1
Peptides having the sequence 764 to 2090 of SEQ ID No. 1
Peptides having the sequence 764 to 2091 of SEQ ID No. 1
Peptides having the sequence 764 to 2092 of SEQ ID No. 1
Peptides having the sequence 764 to 2093 of SEQ ID No. 1
Peptides having the sequence 764 to 2094 of SEQ ID No. 1
Peptides having the sequence 764 to 2095 of SEQ ID No. 1
Peptides having the sequence 764 to 2096 of SEQ ID No. 1
Peptides having the sequence 764 to 2097 of SEQ ID No. 1
Peptides having the sequence 764 to 2098 of SEQ ID No. 1
Peptides having the sequence 764 to 2099 of SEQ ID No. 1
Peptides having the sequence 764 to 2100 of SEQ ID No. 1
Peptides having the sequence 764 to 2101 of SEQ ID No. 1
Peptides having the sequence 764 to 2102 of SEQ ID No. 1
Peptides having the sequence 764 to 2103 of SEQ ID No. 1
Peptides having the sequence 764 to 2104 of SEQ ID No. 1
Peptides having the sequence 764 to 2105 of SEQ ID No. 1
Peptides having the sequence 764 to 2106 of SEQ ID No. 1
Peptides having the sequence 764 to 2107 of SEQ ID No. 1
Peptides having the sequence 764 to 2108 of SEQ ID No. 1
Peptides having the sequence 764 to 2109 of SEQ ID No. 1
Peptides having the sequence 764 to 2110 of SEQ ID No. 1
Peptides having the sequence 764 to 2111 of SEQ ID No. 1
Peptides having the sequence 764 to 2112 of SEQ ID No. 1
Peptides having the sequence 764 to 2113 of SEQ ID No. 1
Peptides having the sequence 764 to 2114 of SEQ ID No. 1
Peptides having the sequence 764 to 2115 of SEQ ID No. 1
Peptides having the sequence 764 to 2116 of SEQ ID No. 1
Peptides having the sequence 764 to 2117 of SEQ ID No. 1
Peptides having the sequence 764 to 2118 of SEQ ID No. 1
Peptides having the sequence 764 to 2119 of SEQ ID No. 1
Peptides having the sequence 764 to 2120 of SEQ ID No. 1
Peptides having the sequence 764 to 2121 of SEQ ID No. 1
Peptides having the sequence 764 to 2122 of SEQ ID No. 1
Peptides having the sequence 764 to 2123 of SEQ ID No. 1
Peptides having the sequence 764 to 2124 of SEQ ID No. 1
Peptides having the sequence 764 to 2125 of SEQ ID No. 1
Peptides having the sequence 764 to 2126 of SEQ ID No. 1
Peptides having the sequence 764 to 2127 of SEQ ID No. 1
Peptides having the sequence 764 to 2128 of SEQ ID No. 1
Peptides having the sequence 764 to 2129 of SEQ ID No. 1
Peptides having the sequence 764 to 2130 of SEQ ID No. 1
Peptides having the sequence 764 to 2131 of SEQ ID No. 1
Peptides having the sequence 764 to 2132 of SEQ ID No. 1
Peptides having the sequence 764 to 2133 of SEQ ID No. 1
Peptides having the sequence 764 to 2134 of SEQ ID No. 1
Peptides having the sequence 764 to 2135 of SEQ ID No. 1.
Peptides having the sequence 764 to 2136 of SEQ ID No. 1
Peptides having the sequence 764 to 2137 of SEQ ID No. 1
Peptides having the sequence 764 to 2138 of SEQ ID No. 1
Peptides having the sequence 764 to 2139 of SEQ ID No. 1
Peptides having the sequence 764 to 2140 of SEQ ID No. 1
Peptides having the sequence 764 to 2141 of SEQ ID No. 1
Peptides having the sequence 764 to 2142 of SEQ ID No. 1
Peptides having the sequence 764 to 2143 of SEQ ID No. 1
Peptides having the sequence 764 to 2144 of SEQ ID No. 1
Peptides having the sequence 764 to 2145 of SEQ ID No. 1
Peptides having the sequence 764 to 2146 of SEQ ID No. 1
Peptides having the sequence 764 to 2147 of SEQ ID No. 1
Peptides having the sequence 764 to 2148 of SEQ ID No. 1
Peptides having the sequence 764 to 2149 of SEQ ID No. 1
Peptides having the sequence 764 to 2150 of SEQ ID No. 1
Peptides having the sequence 764 to 2151 of SEQ ID No. 1
Peptides having the sequence 764 to 2152 of SEQ ID No. 1
Peptides having the sequence 764 to 2153 of SEQ ID No. 1
A further embodiment of the invention is a composition comprising a complex of Factor VIII with one or more Von Willebrand Factor peptides, wherein                the Von Willebrand factor peptides are fragments of Von Willebrand Factor        the complex of Factor VIII and the fragments of Von Willebrand Factor show a reduced binding to phospholipid membranes compared to Factor VIII alone        the complex of Factor VIII and the fragments of Von Willebrand Factor show a reduced binding to collagen III compared to the complex of Factor VIII and full length Von Willebrand Factor        the complex of Factor VIII and the fragments of Von Willebrand Factor show a reduced binding to heparin compared to the complex of Factor VIII and full length Von Willebrand Factor.        
Preferably, the Von Willebrand Factor peptides have a molecular weight <500 kD, preferably <400 kD. As the Von Willebrand Factor often forms oligomers or multimers, also the peptides of the present invention may be in the form of multimers or oligomers.
In a preferred embodiment the peptides of the present invention have at least one property selected from the group consisting of                (i) an affinity binding constant for heparin of KD>1 nM, preferably ≥2.43 nM        (ii) an affinity binding constant for collagen III of KD>5 nM, preferably ≥17.02 nM        (iii) an affinity binding constant for Factor VIII of KD<100 nM or <10 nM, preferably ≤6.19 nM and        (iv) an inhibition of Factor VIII phospolipid binding of at least 70%, preferably at least 80% or at least 90%.        
The Von Willebrand factor peptides of the invention show preferably a reduced binding to heparin, a lower affinity for collagen (like collagen III), a lower affinity to phospholipids but still a high binding to Factor VIII.
Surprisingly, low binding to phospolipids and collagen improves release rate in case of non-intravenous administration, especially subcutaneous.
The measurement of the respective affinity binding constants is described in the experimental part.
In one embodiment, the Von Willebrand Factor peptides are derived from Von Willebrand Factor by proteolytic or chemical cleavage. If proteolytic cleavage is used, S. aureus V-8 protease is especially preferred.
Preferably, the composition of the present invention has at least one of the following properties:                (i) the Von Willebrand Factor peptides shield Factor VIII from antibody binding to minimize inhibitor formation in a patient        (ii) stabilises Factor VIII to provide a remaining Factor VIII activity of at least 90% after storage for 12 month in a frozen liquid form at −70° C.        (iii) stabilises Factor VIII to provide a remaining Factor VIII activity of at least 90% after storage for 24 month in a freeze-dried form at 5° C.        (iv) stabilises Factor VIII to provide a remaining Factor VIII activity of at least 90% after storage for 12 month in a freeze-dried form at 25° C.        (v) prolonges half-life of Factor VIII in-vivo by at least 20% and        (vi) reduces inhibitor formation in previously untreated patients to less than 20%, preferably less than 10% after treatment with the composition for 6 months.        
Surprisingly, the Von Willebrand Factor peptides seem to increase stability of Factor upon storage (shelf-life) and/or reduce inhibitor formation in patients. Inhibitor formation is one of the major problems in the treatment of chronic bleeding disorders.
The composition of the present invention is especially useful in the treatment or prevention of a bleeding disorder.
Therefore, a further embodiment of the invention is a method of treating a bleeding disorder comprising administering to a patient in need thereof an effective amount of the composition of the present invention.
The amount depends on the disease or condition to be treated and may be selected by a person skilled in the art. For long term treatment, amounts of 20 to 40 IU/kg bodyweight per application are typically suitable. In an emergency situation, the amount may be about 10 to 50 IU/kg bodyweight.
The composition of the invention may be applied by intravenous administration or non-intravenous administration. The non-intravenous administration may be a subcutaneous injection, an intradermal injection or an intramuscular administration.
One advantage of the method of the present invention is the possibility to use nano filtration for virus removal. Von Willebrand Factor, because of its size, may not be nanofiltrated with a nanofilter with a small pore size to remove viruses. Because the Von Willebrand Factor peptides are much smaller in size than the full length Von Willebrand Factor molecule, nanofiltration with small pore sizes becomes possible. Nanofiltration is done at a pore size and conditions that reduces the concentration of one of the smallest known viruses porcine parvovirus by a least a factor of 100 (2 log), preferably by at least a factor 1000 (3 log) and most preferably to a concentration below detection limit of the parvovirus assay, optionally using one or more nanofilters in series. For this test, porcine parvovirus is spiked in a sample and analysed after filtration.
Therefore, a further embodiment of the invention is a method for virus reduction comprising the step of nanofiltrating the Von Willebrand Factor peptides prior or after a combination with Factor VIII, whereby porcine parvovirus would be reduced by at least 2 log.
A preferred puffer for administration of the composition of the invention comprises melizitose, preferably in an amount of up to 1,000 mM particularly from about 10 mM to about 200 mM, in particular from about 10 mM to about 100 mM.
A further embodiment of the invention is a method of preparing Von Willebrand Factor peptides comprising the following steps:                Incubating Von Willebrand Factor with S. aureus V-8 protease for 2 to 16 hours at an enzyme to Von Willebrand Factor weight/weight ratio of 1:5 to 1:100        Binding and purifying on an anion exchanger and collecting the desired purified vWF peptides in a fraction coming from the anion exchanger by applying an increased amount of salt concentration.        